This project is designed to evaluate the relationship between the metabolism of myoinositol and its phospholipid derivatives and carbohydrated-stimulated insulin secretion from the rat pancreatic islet. The substrate and ionic requirements for phosphatidylinositol hydrolysis will be determined. The subcellular localization and characteristics of the enzymes responsible for phosphatidylinositol synthesis (CDP-diglyceride:inositol phosphotransferase) and hydrolysis (phosphatidylinositol phosphohydrolase) will be studied. Electron microscopic radioautography will be employed to determine the subcellular localization and the pattern of incorporation of radioactive myoinositol into the membrane phospholipids of the beta-cell and the changes which follow exposure of the pancreatic islet to stimulatory conditions (carbohydrates, amino acids, and calcium ionophores). Methods will be developed to permit the isolation of insulin granules and plasma membranes from catfish islets as well as to isolate well-characterized insulin granule populations by isoelectric focusing. These preparations will be utilized to determine the relationship between the metabolism of phosphatidylinositol by the limiting membrane of the insulin granule and its ability to fuse with the plasma membrane of the beta-cell. Once these relationships have been established, it may be possible to determine whether defects in the metabolism of phosphatidyl-inositol are involved in the abnormal secretion of insulin associated with diabetes mellitus.